The development of coronary heart disease (CHD) involves phenotypic changes in resident vascular smooth muscle cells (VSMCs) that contribute to plaque growth and inflammation at the site of injury. During this process, contractile VSMCs within the medial layer of the blood vessel become activated, taking on a synthetic phenotype that persists throughout the lifetime of the disease. Although synthetic VSMCs are capable of reverting to the healthy contractile phenotype, the factors governing this transition, as well as the full role of synthetic VSMCs in CHD development, remain insufficiently understood. TCF21 is one of a class of transcription factors that regulate organ development and cell differentiation and several SNPs within the TCF21 locus have been recently identified as risk factors for CHD development. Also, preliminary evidence strongly implicates TCF21 as a key regulator of VSMC behavior, but the mechanisms of its influence are poorly understood. Consequently, TCF21 is a pertinent target for further investigation into the causes and dynamics of VSMC pathology. The overall goal of the proposed project is to define the genetic programming driven by TCF21 activity during the VSMC transition between the contractile and synthetic phenotypes. Three Specific Aims make up the investigative strategy: 1) Identify regions of the human VSMC genome that directly interact with TCF21; 2) Determine the effect of TCF21 on VSMC gene expression; 3) Identify genetic networks and enriched genes and pathways regulated by TCF21. Aim 1 will be accomplished with Chip-Seq assays on human coronary artery smooth muscle cells (HCASMCs) natively expressing TCF21 that are cultured in conditions stimulating either the contractile or synthetic phenotype. Aim 2 will apply RNA-Seq to contractile or synthetic HCASMCs treated with control or anti-TCF21 siRNA, which reduces TCF21 expression to less than 30% of controls. Aim 3 will apply sophisticated computational methods to data from Aims 1 and 2 to define a set of TCF21 core genes and TCF21 differentiation genes, and infer the structure of genetic networks regulated by TCF21 by mining existing gene ontology databases including GO, KEGG, and Ingenuity. Collectively, conclusions from the proposed studies will inform ongoing investigation of the genetic basis of coronary heart disease and be of great interest to fields concerned with vascular smooth muscle cell biology, cardiovascular disease, and transcriptional regulation of phenotypic plasticity in various cell types.